The goals of this proposal are to examine the regulation and function of the alpha subunit of the amiloride-sensitive epithelial Na+ channel (ENaC). The activity of this channel determines the extent of Na+ reabsorption and K+ secretion in the distal nephron. Aldosterone, glucocorticoids and cAMP have important regulatory influences on channel activity. We have recently identified and characterized multiple human alphaENaC (halphaENaC) transcripts that are regulated by distinct promoters and encode two functional N-terminal variant proteins. This proposal has two broad goals: The first is to examine the regulation of halphaENaC isoforms in renal and airway epithelial cells. We hypothesize that corticosteroids and cAMP regulate amiloride-sensitive Na+ transport by transcriptional regulation of alphaENaC expression. The second is to examine the role of the N-terminus of alphaENaC in protein-protein interactions and in conferring functional properties to the ENaC complex. We hypothesize that functional heterogeneity of ENaC transporting epithelia may be in part from molecular variants of the ENaC complex and that cytosolic effectors interact differently with each of the N-terminal isoforms of alphaENaC. The data presented with this proposal includes the identification and characterization of human alphaENaC alternate transcripts and their transcriptional regulation by corticosteroids in renal and lung cell lines. A functional hormone response element in the 5' flanking region of halphaENaC is discovered and variant alphaENaC proteins are shown to reconstitute a functional amiloride- sensitive ion channel. The specific aims are: (1) to examine transcriptional regulation of alphaENaC gene expression in kidney collecting duct and airway epithelia; (2) to characterize transcription factors that regulate alphaENaC expression; and (3) to determine the role of the N-terminus of alphaENaC in protein- protein interactions and in conferring functional properties to the ENaC complex. The long-term objectives are to understand the regulation and function of ENaC in transepithelial Na+ transport.